This invention pertains to proteins required for activated transcription in fungi, nucleic acids encoding these proteins, and methods for using these proteins.
Most fungi are opportunistic pathogens, producing serious disease only in compromised individuals. As the result of an aging population as well as an increase in the number of immunocompromised patients, e.g., patients with acquired immunodeficiency syndrome (AIDS), patients undergoing cancer chemotherapy, or immunosuppressive therapy, e.g., treatment with corticosteroids, and patients undergoing organ transplantation, fungal infections are increasing rapidly.
Most infections begin by colonization of either the skin, a mucosal membrane, or the respiratory epithelium. Passage through the initial surface barrier is accomplished through a mechanical break in the epithelium. Although most fungi are readily killed by neutrophils, some species are resistant to phagocytic killing and can infect otherwise healthy individuals.
Fungi parasitize many different tissues. Superficial fungi cause indolent lesions of the skin. Subcutaneous pathogens cause infection through the skin and spread by subcutaneous or lymphatic routes. Opportunistic fungi such as Aspergillus are widespread in the environment and are present in normal flora. Fungi cause disease primarily in immunocompromised individuals. Systemic fungi are the most virulent and may cause progressive disease leading to deep seated visceral infections in otherwise healthy individuals (see e.g. Sherris Medical Microbiology, Third Edition, Kenneth J. Ryan, ed., Appleton and Lange, Norwalk, Conn., 1994).
The major fungal pathogens in North America are Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Cryptococcus neoformans, Candida species, such as but not limited to Candida albicans and Aspergillus species (Medically Important Fungi, Second Edition, Davise H. Larone, Ed., American Society for Microbiology, Washington, D.C.). C. albicans is the most frequent cause of candidiasis; symptoms range from an acute to chronic infection involving any part of the body.
Fungi are a distinct class of microorganism, most of which are free-living. They are eukaryotic organisms containing a nuclear membrane, mitochondria and an endoplasmic reticulum. The cell structure includes a rigid cell wall of mannan, glucan, and chitin and a cytoplasmic membrane with a large percentage of ergosterol. The size and morphology of fungi vary. Candida are monomorphic yeasts and yeast-like organisms including Candida, Cryptococcus, and Saccharomyces.
Only a handful of agents are active against fungi. For life threatening disease caused by any of these fungi, amphotericin B is the agent of choice. This drug, however, is associated with numerous severe side effects such as fever, dyspnea and tachycardia, and dosage is limited over the lifetime of the patient because of renal toxicity. An agent frequently used concurrently is flucytosine, a nucleoside analog, which cannot be used independently of other agents because of the rapid appearance of resistance. Untoward effects of treatment with flucytosine include leukopenia, thrombocytopenia, rash, nausea, vomiting, diarrhea, and severe enterocolitis.
In conditions where the patient""s life is not threatened, ketoconazole can be used as a long-term therapy for blastomycosis, histoplasmosis, or coccidioidomycosis. Fluconazole also has a significant role in the treatment of superficial fungal infections. Both compounds are from the same class, the triazoles, and are cytostatic. The emergence of resistance and hepatic toxicity limits the use of triazoles such as fluconazole and ketoconazole. The newest triazole, itraconazole, has similar pharmacokinetics and spectrum of activity as fluconazole. None of the azoles can be used for life threatening or deep seated fungal infections. They are only effective in reducing colonization of fungi such as Candida and for treating superficial mycoses.
All major antifungal agents attack directly or indirectly a component of the cell wallxe2x80x94ergosterol. Amphotericin B and other polyene macrolides interact with ergosterol in the cell membrane and form pores or channels that increase the permeability of the membrane. Resistant to amphotericin B in mutant strains is accompanied by decreased concentrations of ergosterol in their cell membranes. Imidazoles and triazoles inhibit sterol 14-xcex1-demethylase, a microsomal cytochrome P450-dependent enzyme system. Imidazoles and triazoles thus impair the biosythesis of ergosterol for the cytoplasmic membrane, leading to the accumulation of 14-xcex1-methyl sterols, which impair certain membrane-bound enzyme systems (See, The Pharmacological Basis of Therapeutics, Eighth Edition, Goodman and Gilman, Pergamon Press, 1990).
Development of an effective method and composition for treatment of fungal infections is a critical goal of the pharmaceutical industry. The pharmaceutical industry has made numerous efforts to identify fungal-specific drugs, with only limited success to date. It would be of great value to identify a new class of antifungal drugs that block a fungal target other than ergosterol. This target should be fungal-specific and should lead to development of a drug that is effective against the organisms that are resistant to current therapy.
Drug development often relies on the screening of a large number of potential inhibitors before a specific lead compound inhibitor is found. Assays developed for such screens are complex and must mimic the physiological activity of the target protein. Thus, it is critical for the development of these screens to define the proteins involved in the targeted process and to have discovered a means of purifying the necessary components of the assay. In addition, it is useful to have clones for the protein components of the assay to facilitate the production of these fungal specific components.
Therefore, there is a need in the art to identify one or more fungal constituents, preferably polypeptides, that can serve as useful targets for drug intervention, and for methods and compositions for identifying useful anti-fungal agents and treating fungal infections.
The present invention relates to components of the transcriptional apparatus in C. albicans. The RNA polymerase II holoenzyme includes (i) subunits of RNA polymerase needed for some or all of the stages of transcription, but which are not specific for individual promoters and (ii) general transcription factors, termed SRBs, which bind RNA polymerase and regulate initiation of transcription at all promoters. In addition, specific transcription factors, which bind to specific sequences in particular promoters, further regulate the transcriptional activity of RNA polymerase II. All of these factors represent potential targets for antifungal agents.
The present invention is based on the isolation of nucleic acid encoding Candida albicans SRB-7 (CaSRB-7), which forms part of the RNA polymerase II holoenzyme complex. In one aspect, the invention provides an isolated nucleic acid having the sequence depicted in FIG. 2, SEQ ID NO:1, as well as sequence-conservative and function-conservative variants thereof. The invention also provides vectors comprising these sequences, and cells comprising the vectors. Methods for producing the polypeptides, which comprise (i) culturing the cells and (ii) recovering the polypeptide from the culture, are also provided.
In another aspect, the invention provides an isolated polypeptide having the amino acid sequence depicted in FIG. 2, SEQ ID NO:2, and function-conservative variants thereof.